Dilute Filtration Sterilization Process for Viscoelastic Biopolymers

ABSTRACT

Manufactured hyaluronic acid products are used in numerous surgical applications including viscoelastic supplementation for the treatment of osteoarthritis; however, traditional sterilization techniques result in the breakdown of such high molecular o weight viscoelastic biopolymers and are thus unsuitable. Disclosed are processes for obtaining concentrated sterile solutions of high molecular weight biopolymers such as hyaluronic acid. The processes include filter sterilization with a dilute preparation of the biopolymer, and concentration of the dilute filter sterilized biopolymer by ultrafiltration to a desired concentration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/023,196, which was filed on Sep. 10, 2013 which, in turn, is acontinuation-in-part of U.S. application Ser. No. 12/742,861, which wasfiled on Dec. 13, 2010 as the national phase under 35 U.S.C. 371 of PCTInternational Appl. PCT/IB2008/003042, filed on Nov. 12, 2008, whichclaims the benefit of European Patent Appl. 07120568.6, filed on Nov.13, 2007. The subject matter of each of these prior applications isincorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a process for formulating a sterileviscoelastic biopolymer such as hyaluronic acid, following bulkmanufacture.

BACKGROUND OF THE INVENTION

The invention relates to methods of formulation of viscoelasticbiopolymers following bulk manufacture. The biopolymers to which theinvention may be applied include homopolysaccharides andheteropolysaccharides, particularly the category ofheteropolysaccharides known as glycosaminoglycans. Glycosaminoglycansespecially suitable for use in the present invention are hyaluronic acid(HA), chondroitin sulfate, dermatan sulfate, keratan sulfate, heparinand heparan sulfate.

HA is a naturally occurring biopolymer consisting of repeatingdisaccharide units of D-glucuronic acid in β-(1-3) linkage withN-acetyl-D-glucosamine, wherein each disaccharide unit is connected toits adjoining neighbors by β-(1-4) linkages. The salt sodium hyaluronate(NaHA) is found at physiological pH in human and vertebrate jointsynovial fluid, connective tissue, vitreous humor of the eye and healthyskin tissue, and is an extracellular secretion product of severalbacterial species, particularly of the genus Streptococcus.

To date, the major medical applications of manufactured NaHA productsare in ophthalmic surgery for cataracts and intraocular lensimplantation, in dermatological applications for filling wrinkles andaugmenting lip size, and in viscoelastic supplementation for thetreatment of osteoarthritis in humans and large mammals. Viscoelasticsupplementation, particularly in the knee, is aimed at restoring thenormal rheological homeostasis of the joint network and for providingimmediate protection, lubrication, shock absorption, hydrodynamicresistance and a mechanochemical barrier against stress. Intra-articularinjections of NaHA have been shown to improve function and mobility anddecrease pain.

More recent medical applications of manufactured NaHA include medicaldevice coatings, surgical adhesion prevention products, drug deliveryvehicles, bone replacement materials and wound healing materials.

Marketed NaHA products include BioLon™, Biolon™ Prime, BioHy™ (all fromBiotechnology General (Israel) Ltd.), Hyalart™ (Fidia), Synvisc™ HyalanG-F 20 (Biomatrix), Healon™ (Pharmacia), BD Vise™ (Becton Dickinson) andOrthovisc™ (Anika Therapeutics).

For commercial applications in the pharmaceutical, cosmetic and foodindustries, the quality of a viscoelastic biopolymer is dependent on thecombined parameters of viscosity, concentration and molecular weight.For example, HA for pharmaceutical use must be of high molecular weightto ensure sufficient water retention, yet the viscosity must be ofreasonable order so as to enable ease of administration andmanipulation, e.g., syringeability. NaHA found in biological sourcessuch as rooster combs and culture broths of fermented Streptococcusstrains, is often of very high molecular weight, i.e., >3×10⁶ daltons.Procedures used for its extraction, purification and sterilizationtypically result however, in a final product in which the molecularweight is significantly reduced as compared to the native compound.

For example, lyophilization of NaHA following repeated extractioninduces sublimation of water, the net result of which is shearing ofhigh molecular weight molecules. Low pH techniques result in formationof cross-links, which break upon subsequent pH increase and contributeto shearing of NaHA.

Sterilization techniques, such as those employing dry or moist heat,liquid chemicals, ethylene oxide gas, UV radiation, electron beanradiation, gamma radiation, microwaves and ultrasound all result inbreakage of linear molecules, and are thus unsuitable for a viscoelasticbiopolymer product in which molecular weight is a key parameter foroptimal product quality.

NaHA manufacture from biological sources is well known to those skilledin the art. A typical bulk purification process (of which there are manyvariations) involves repeated extraction, precipitation, absorption,centrifugation and/or filtration steps to remove contaminants.Procedures for isolation of NaHA from fermented Streptococcus culturesare disclosed for example, in U.S. Pat. No. 4,780,414 and U.S. Pat. No.4,784,990 (both assigned to BioTechnology General (Israel) Ltd.), U.S.Pat. No. 5,563,051, U.S. Pat. No. 5,411,874 (Fermentech Medical), U.S.Pat. No. 5,071,751 (assignee Chisso Corp.) and U.S. Pat. No. 5,316,916.

U.S. Pat. No. 5,023,175 (assignee Kabushiki) relates to purification ofcosmetic grade NaHA (MW 2.1×10⁶) with dialysis ultrafiltration as thefinal step prior to freeze drying.

U.S. Publication No. 2002/0120132 relates to purification of NaHA(MW>7.5×10⁵) involving a temperature controlled reactor and allegedlyless ethanol than traditional methods. The purified NaHA is dried undervacuum or lyophilized.

Alternate methods for bulk manufacture of NaHA from Streptococcalcultures avoid extraction/precipitation steps, and rely primarily onsequential filtration techniques as disclosed for example, in GB2,249,315 (assignee Chisso Corp.), WO 95/04132 (applicant Fidia Corp.)and U.S. Pat. No. 6,489,467 (assignee Chemedica).

None of the above disclose processes for formulating a highly purifiedbulk manufactured viscoelastic biopolymer such as NaHA into a finalsterile product suitable for medicinal use.

U.S. Pat. No. 4,141,973 (assignee Biotrics) discloses a sterile HAproduced by extensive purification of material from rooster combs anddissolution of the final product in sterile phosphate buffered saline.The formulation however, has measurable amounts of protein and otherimpurities.

U.S. Pat. No. 4,517,295 (assignee Diagnostic Inc.) discloses aStreptococcal NaHA product prepared by a process in whichsterile-filtration is the terminal step. The disclosed method suffersfrom the disadvantage of producing low molecular weight NaHA (average MW5.5×10⁴ daltons).

U.S. Pat. No. 5,093,487 (assignee Mobay Corp.) and U.S. Pat. No.5,316,926 (assignee Miles Inc.) disclose final filter-sterilization of aStreptococcal NaHA formulation (average MW 1-2×10⁶ daltons), following apurification method comprising a mechanical winding technique. Thepurpose of the winding technique is to increase both molecular weightand viscosity, after which heat treatment or filtration is performedallegedly to reduce viscosity without affecting molecular weight. Thewinding method is disadvantageous for its unknown effects on thechemical composition of NaHA.

U.S. Pat. No. 4,782,046 (assignee Mobay Corp.) relates to preparing afinal NaHA product (average MW generally less than 3.0×10⁶ daltons) byfilter sterilization and/or beta-propiolactone treatment prior tosyringe filling. Beta-propiolactone treatment is disadvantageous astraces may remain in the preparation following hydrolization, and it mayadversely affect the chemical structure of NaHA.

U.S. Pat. No. 5,079,236 (assignee Hyal Pharmaceutical Corp.) relates topreparing a formulation by dissolving purified NaHA (average MW5-20×10⁴), optionally containing a steroid, in a heated solution ofpreservatives, e.g., sodium benzoate, methylparaben and propylparaben,adjusting the pH and volume, filling vials and sterilizing the vials inan autoclave. Autoclave sterilization is deleterious to NaHA molecularweight.

U.S. Pat. No. 5,411,874 and U.S. Pat. No. 5,563,051 (assigneeFermentech) relate to preparing a medical grade NaHA solution bydissolving NaHA (average MW 1.6-2.5×10⁶) purified by repeatedprecipitations in sterile phosphate buffered saline.

WO 01/28602 (applicants Genetics Institute and Fidia) relates toinjectable formulations comprising HA esters and osteogenic protein.

U.S. Pat. No. 6,221,854 (assignee Orquest) relates to injectionformulations comprising NaHA and growth factors.

None of the above disclose industrially applicable methods forformulating bulk manufactured and purified viscoelastic biopolymer suchas NaHA into a final product suitable for medicinal use.

An object of the invention is to provide a method for formulating a bulkmanufactured and purified viscoelastic biopolymer such as NaHA into afinal product suitable for medicinal use.

An object of the invention is to produce a formulation comprising highmolecular weight viscoelastic HA that is suitable for administration byinjection into ocular and intra-articular spaces in humans and animals.

An object of the invention is to provide an industrial process forformulating NaHA obtained by bulk manufacture into a final product ofaverage molecular weight 3×10⁶ or greater suitable for medicinal usewithout subjecting the NaHA to freeze-drying at any stage of manufactureor formulation.

SUMMARY OF THE INVENTION

The invention provides a process for formulating a viscoelasticbiopolymer comprising the steps of:

-   -   i. sterile-filtering soluble bulk manufactured biopolymer by        passage through a membrane suitable for sterile filtration; and    -   ii. concentrating the biopolymer by ultrafiltration until a        desired final concentration is obtained.

The invention also provides a process for formulating a viscoelasticbiopolymer comprising the steps of:

-   -   i. dissolving bulk manufactured biopolymer in a suitable buffer        medium to at least a concentration dilute enough to be suitable        for sterile-filtering;    -   ii. sterile-filtering the biopolymer by passage through a        membrane suitable for sterile filtration; and    -   iii. concentrating the biopolymer by ultrafiltration until a        desired final concentration is obtained.

The invention also provides a process for formulating a viscoelasticpreparation of hyaluronic acid comprising the steps of:

-   -   i. dissolving bulk manufactured hyaluronic acid in a suitable        buffer medium to at least a concentration dilute enough to be        suitable for sterile-filtering;    -   ii. sterile-filtering the dissolved hyaluronic acid by passage        through a 0.2 micron absolute membrane; and    -   iii. concentrating the hyaluronic acid by ultrafiltration until        a desired final concentration is obtained.

In an additional embodiment the viscoelastic biopolymer is not subjectedto freeze drying at any stage of bulk manufacture or formulation.

The invention is suitable for use with glycosaminoglycan biopolymerssuch as HA. The invention is most suitable for use with viscoelasticbiopolymers of high molecular weight, for example HA having a molecularweight in the range of 1×10⁴ to 1×10⁷ daltons and having apseudoplasticity index in the range of 600 to 1200.

The formulations provided by the invention are highly purified, sterileand have favorable rheological properties and are in a form appropriatefor injection into ocular and intra-articular spaces in humans andanimals.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a process for formulating a viscoelasticbiopolymer comprising the steps of:

-   -   i. dissolving bulk manufactured viscoelastic biopolymer in a        suitable medium to at least a concentration dilute enough to be        suitable for sterile-filtering;    -   ii. sterile-filtering the dissolved viscoelastic biopolymer by        passage through a membrane suitable for sterile filtration; and    -   iii. concentrating the sterile-filtered viscoelastic biopolymer        by ultrafiltration until a desired final concentration is        obtained.

The method of the invention is suitable for use with viscoelasticbiopolymers for which a highly purified and sterile formulation isrequired. The viscoelastic biopolymer may be within a wide range ofmolecular weight, e.g., 1×10⁴ to 1×10⁷ daltons, but the method is mostparticularly suitable for use with viscoelastic biopolymers of highmolecular weight since viscosity is a function of both concentration andmolecular weight. According to the method of the invention, a bulkmanufactured and highly purified viscoelastic biopolymer issterile-filtered at a relatively low concentration to enable itsefficient passage through the filtration apparatus at an appliedpressure which does not contribute to biopolymer shearing. As theterminal step in the formulation process prior to package or devicefilling, the sterile biopolymer is concentrated by ultrafiltration to adesired final concentration.

As used herein the term “high molecular weight” depends on theparticular biopolymer to be formulated but generally refers to amolecular weight greater than 1×10⁶ daltons. A high molecular weight HAviscoelastic biopolymer is generally in the range of 1×10⁶ to 1×10⁷daltons, and more particularly in the range of 2.5×10⁶ to 5.0×10⁶daltons. The method is also applicable to viscoelastic biopolymerpreparations in which the molecular weight has been intentionallyreduced. For example, reduced molecular weight preparations of HA may beobtained following irradiation treatment, as described in U.S. Pat. No.6,383,344, or by treatment with ultrasound and sodium hypochlorite asdescribed in U.S. Pat. No. 6,232,303. Other high molecular weights of

HA viscoelastic biopolymer useful in the method of the invention may beat least about 1×10⁶ daltons, at least about 1×10⁷ daltons, or at leastabout 1×10⁸ daltons.

Still other high molecular weight HA viscoelastic biopolymers useful inthe method of the invention may be no more than about 2.5×10⁶ daltons,no more than about 5.0×10⁶ daltons, no more than about 1×10⁷ daltons, orno more than about 1×10⁸ daltons. Alternatively, the high molecularweight HA viscoelastic biopolymers may be about 1×10⁶ to about 2.5×10⁶daltons, about 1×10⁶ to about 5.0×10⁶ daltons, about 1×10⁶ to about1×10⁷ daltons, about 1×10⁶ to about 1×10⁸ daltons, about 2.5×10⁶ toabout 5.0×10⁶ daltons, about 2.5×10⁶ to about 1×10⁷ daltons, about2.5×10⁶ to about 1×10⁸ daltons, about 5.0×10⁶ to about 1×10⁷ daltons,about 5.0×10⁶ to about 1×10⁸ daltons, or about 1×10⁷ to about 1×10⁸daltons.

The viscoelastic biopolymer suitable for the method of the invention maybe a homopolysaccharide, i.e., assembled from a single type ofmonosaccharide, or a heteropolysaccharide, i.e., assembled from two ormore different types of monosaccharides. Examples of homopolysaccharidesinclude carboxymethylcellulose, chitin, polymannuronic acid, curdlangum, scleroglucan and dextran. Examples of heteropolysaccharides includeglycosaminoglycans, alginates, carageenans, guar gum, pectins, locustbean gum and xanthum gum. The method of the invention is particularlysuitable for glycosaminoglycans, also known as acid mucopolysaccharides,which are composed of repeating disaccharide units in which one of thetwo monosaccharides is always either N-acetylglucosamine orN-acetylgalactosamine. Examples of glycosaminoglycans include HA,chondroitin, chondroitin sulfate A, chondroitin sulfate B, chondroitinsulfate C, dermatan sulfate, keratan sulfate, heparin and heparansulfate. Both homo- and heteropolysaccharides may be linear or branchedstructures. The component monosaccharides of a biopolymer may bereleased by acid hydrolysis and detected by analytical techniques suchas thin layer chromatography and/or high pressure liquid chromatography.

The viscoelastic biopolymer should be soluble in aqueous solution. Thosewhich are soluble and liquid at temperatures in the range of 10-30° C.are most suitable for the method of the invention, but those which aresoluble only at elevated temperatures e.g., locust bean gum may also beformulated using the method of the invention. A viscoelastic biopolymerwhich is normally insoluble in aqueous solution may be rendered suitablefor the method of the invention upon derivatization by chemicaltreatment. For example, while cellulose is insoluble, the derivativecarboxymethylcellulose formed by reaction of cellulose with alkali andchloroacetic acid, is soluble and may be formulated using the method ofthe invention.

According to the invention, the biopolymer may be in its native form orit may be chemically modified and/or derivatized. Examples of chemicalmodification/derivatization include cross-linking (U.S. Pat. No.6,552,184), addition of sulfate (WO 95/25751; WO 98/45335), carboxyl orhydroxyl groups, attachment of lipophilic side chains, introduction ofacetyl groups, and esterification with and without additional moietiesattached (EP Pat. No. 216,453; WO 98/08876). Additional moieties includedrugs, polysaccharides, lectins, imaging agents, targeting proteins suchas antibodies, growth factors, and the like.

The native form of a biopolymer may an anionic, cationic or neutral saltform.

The method of the invention is particularly suitable for HA. The termhyaluronic acid (HA) means hyaluronic acid, salts thereof, such assodium, potassium, magnesium, calcium, lysine, ammonium, triethanolamineand propanolamine hyaluronates, metal salts thereof, such as cobalt,zinc, copper, iron, manganese and lithium hyaluronate, and chemicallymodified and derivatized forms thereof, as disclosed for example in U.S.Pat. No. 4,851,521, U.S. Pat. No. 5,099,013, U.S. Pat. No. 5,336,767,and U.S. Pat. No. 6,017,901.

As used herein, the term viscoelastic refers to the rheological behaviorof a biopolymer solution, which under the effect of shear displays boththe characteristics of a purely elastic material, i.e., capable ofstoring energy, and the characteristics of a purely viscous material,i.e., capable of dissipating energy.

Rheological behavior is characteristic and specific and is a function ofthe biopolymer's length, structure and charge. Some biopolymers, such asHA, display non-Newtonian behavior, indicating that the viscosity isdependent on both shear rate and temperature, and they displaypseudoplastic behavior (also known as “shear-thinning”), which meansthat the solution viscosity decreases as a function of increasing shearforce.

Viscosity of a particular biopolymer is quantified at a set of discreteshear rates and temperatures, e.g., using a Brookfield viscometer.Further information may be obtained over a continuous range of shear,e.g., using a Haake rotational viscometer.

Viscoelasticity may be quantitated as the ratio of the viscosity at alow shear rate, e.g., 0.1 sec⁻¹, to the viscosity at a high shear rate,e.g., 1000 sec⁻¹. As used herein, viscoelasticity refers to the ratio ofmeasured viscosity at an applied shear rate of 0.1 sec⁻¹ to the measuredviscosity at an applied shear rate of 1000 sec⁻¹. This ratio is alsoknown as the pseudoplasticity index (PI):

PI=viscosity at shear rate 0.1 sec⁻¹/viscosity at shear rate 1000 sec⁻¹

As used herein, a viscoelastic biopolymer has a pseudoplasticity indexgreater than about 500. The PI is a useful index for characterizing thebehavior of a biopolymer under different conditions, and for comparingrheological quality among different biopolymer solutions.

A biopolymer such as HA which is used for viscoelastic supplementationand is administered by intra-articular injection, requires highviscosity at low shear rates, so that it can serve as a non-flowingsupport following injection, but it also requires low viscosity at highshear rate, i.e., while it is being delivered, e.g., by injection (e.g.,through a syringe and needle), so that the delivery can be reasonableeffortless and accurate. Hence the PI of a biopolymer solution directlyinfluences its syringeability.

Syringeability is the force required to expel a biopolymer solution froma syringe or a syringe-like application device. Syringeability may betested using a Force indicator e.g., Mecmesin Force Indicator, and isexpressed in units of g (gravity). For example, a force of 200 g allowsa medical practitioner to efficiently expel NaHA solution from a syringeand simultaneously control the amount of material introduced to thedesired location, e.g., an intra-articular or ocular space.

According to the invention, the biopolymer may be obtained from abiological source, or it may be a product of in vitro enzymatic orchemical synthesis, or combinations thereof. A biological source may bea bacterial, yeast, plant, amphibian, avian or mammalian organism. Forexample, HA may be isolated from any of fermented Streptococcalcultures, human umbilical cords, bovine cartilage or rooster combs. Invitro enzymatic synthesis of polysaccharides, including HA, cellulose,polymannuronic acid and chitin is described in WO 95/24497. The use of arecombinantly-produced hyaluronan synthase enzyme for in vitro synthesisof HA is described in U.S. Pat. No. 6,602,693. HA obtained from any ofthe aforementioned sources may be subsequently chemically modified orderivatized as described, for example, in U.S. Pat. No. 4,851,521, U.S.Pat. No. 4,713,448, U.S. Pat. No. 5,336,767 and U.S. Pat. No. 5,099,013.

Additional viscoelastic biopolymers which are the products of bacterialfermentation include, for example, curdlan gum (β-1-3-D-glucan),produced by Alcaligenes faecalis, gellan gum (tetrasaccharide backboneof L-rhamnose and D-glucose with glyceryl and acetyl substituents)produced by Sphingomonas elodea and xanthum gum (β-1-4-D-glucan withmannose and glucuronic acid side chains), produced by Xanthomonascampedis.

In the field of HA manufacturing, the upstream process of bulkpurification has many variations which are well known to those skilledin the art. Processes for bulk manufacturing of a solution containinghigh molecular weight HA from fermented Streptococcus cultures aredisclosed, for example, in U.S. Pat. No. 5,563,051 and U.S. Pat. No.5,316,916. Other bulk manufacturing processes have been described whichyield solid forms of HA as a final product, as described in U.S. Pat.No. 4,780,414.

The downstream process of formulation, however, is less variable, inthat the usual practice is to sequentially perform concentration andsterilization as the final sequence of operations prior to package ordevice filling. Indeed, according to usual best practice in thebiopharmaceutical industry, sterilization is the final step prior tofilling, and intermediate steps are specifically avoided to minimize thepotential for contamination.

In the process of the invention, the usual industrial practice isunexpectedly avoided and indeed reversed, in that sterilization (byfiltration through an absolute filter of pore size 0.22 micron or less)is carried out prior to concentration (by ultrafiltration). Theconcentration step is carried out aseptically and the final product maybe expected to be of pharmaceutical grade of the highest standardwithout any compromise in sterility, or other quality parameters.Indeed, it is unexpected that an HA product of high molecular weight andviscosity can be formulated on a large aseptic scale where theconcentration step is carried out as an intermediate step betweensterile filtering and filling.

This unexpected order of operations is highly advantageous when appliedto a high molecular weight viscoelastic biopolymer such as HA. One suchsignificant aspect is that the bulk manufactured and dissolved HA (e.g.,0.1%; 1 mg/mL) easily passes through the sterile filter at anindustrially applicable level of pressure to achieve an acceptable flowrate (e.g., minimum flow rate 750 mL/min). The pressure exerted to drivethe HA solution through the sterile filter is sufficiently mild so as tonot adversely affect the molecular weight of the HA.

The reverse process of concentrating e.g., to 1% or 10 mg/mL, followedby terminal sterilization is, in practice, unworkable with a highmolecular weight viscoelastic biopolymer, since the high biopolymerconcentration results in repeated blockage of the sterilizing filterapparatus, resulting in an ultimately inefficient and wastefulprocedure.

Furthermore, using the method of the invention, the bulk manufacturedand filter-sterilized HA is a flexible intermediate product in that itmay be brought to different final concentrations and/or mixed withadditional sterile excipients or active ingredients for different finalformulations.

According to the method of the invention, sterilization of theviscoelastic biopolymer must be carried out by filtration through anappropriate membrane in order to retain the high molecular weightstructure of the polymer. Alternate sterilization techniques, such asthose employing dry or moist heat, liquid chemicals, ethylene oxide gas,UV radiation, electron bean radiation, gamma radiation, microwaves orultrasound result in breakage of long linear molecules, such as those ofHA.

A preferable bulk manufacturing process suitable for the invention isone which consistently yields a viscoelastic biopolymer product which ishomogeneous on a batch to batch basis with respect to a wide range ofphysicochemical and purity parameters. The bulk manufacturing process isessentially a purification process which must be sufficiently rigorousso as to remove minute quantities of impurities originating from theproduction source (e.g., bacterial culture) and from the extractionreagents (e.g., ethanol or cetylpyridinium chloride), all of which cancause adverse reactions if administered to patients. For a product suchas HA intended for injection into patients, the manufacture,specifications and characterization of the bulk manufactured HA shouldbe in accordance with internationally recognized standards andguidelines for the evaluation of toxicity, endotoxin levels andsterility.

On the other hand, the bulk manufacturing process should not comprisesteps which result in excessive shearing of the HA molecule andconcomitantly reduce its molecular weight, e.g., to less than about3×10⁶ daltons, or viscosity, or otherwise result in deviations from thecharacteristic properties of the HA molecule.

A suitable bulk manufacturing process is disclosed for example in U.S.Pat. No. 4,780,414. Such a process comprises the following steps:

-   -   i. precipitating with ethanol a culture broth of a non-hemolytic        nonpathogenic hyaluronic acid-producing fermented Streptococcus        strain;    -   ii. dissolving the precipitate obtained in step (i) in sodium        chloride/ethanol/charcoal;    -   iii. precipitating the dissolved material obtained in step (ii)        with cetylpyridinium chloride;    -   iv. dissolving the precipitate obtained in step (iii) in sodium        chloride/ethanol;    -   v. treating the dissolved material obtained in step (iv) with        magnesium silicate;    -   vi. filtering the treated material obtained in step (v) through        a 0.65 micron absolute membrane; and    -   vii. precipitating the filtrate obtained in step (vi) with        ethanol.

Bulk manufacture and formulation should be carried out only on culturebroth batches in which the molecular weight of HA exceeds a desiredvalue for high molecular weight HA, e.g., 3.0±0.6 megadaltons. Themolecular weight can be in the range from about 2.8 megadaltons to about3.2 megadaltons.

Preferably, the bulk manufacturing process should yield a product inwhich the bioburden is zero or substantially close to zero. Preferablythe zero or substantially close to zero bioburden should be achievedabout half way through the bulk manufacturing process e.g., from thesecond dissolution step of the above-mentioned process. The precipitatedHA is preferably stored under ethanol, and then vacuum dried and storedat 4° C. in sterile containers to protect the bulk manufactured HAagainst contamination prior to the formulation process.

The bulk manufactured HA should be characterized by assessment ofpurity, molecular weight, viscosity, pH, specific rotation,concentration,% HA content and any other necessary parameters in orderto verify batch to batch consistency and quality and thereby assess theefficiency and suitability of the bulk manufacturing process. Preferablythe purity of the bulk manufactured HA is such that the endotoxincontent is <0.25 EU/mL, and more preferably <0.10 EU/mL, and the proteincontent is <1 mg/g, absorbance at 257 nm of a 1% solution is <0.20, theoxidative burst absorbance at 550 nm is <0.10 and the viable count ofaerobic bacteria is <4 CFU/g. Preferably the quality of the bulkmanufactured HA is such that the pH is from 6.0-8.0, the specificrotation is from −72.8°-90.8°, the limiting viscosity number is2680-3410 mL/g, and the molecular weight is 2.4-3.6×10⁶ daltons.

Since HA does not significantly absorb at wavelengths above 240 nm, anysignificant absorbance in the 240-300 nm range is attributable toorganic contaminants such as proteins and nucleic acids. Absorbance at257 nm indicates contamination with nucleotides, DNA or RNA whileabsorbance at 280 nm indicates contamination with proteins or aminoacids. Absorbance at 257 nm below certain limits, e.g., 0.2, isconveniently used as an indication of HA purity. Measured absorbancewhich is substantially close to zero, e.g., <0.08, indicates absolutepurity.

Purity assessment additionally involves analysis for purificationreagents, e.g., ethanol, detergent, used in the bulk manufacturingprocess. Such analyses are conveniently carried out by HPLC and shouldindicate the substantial absence of reagents.

Bacterial endotoxin (lipopolysaccharide) may be quantitated, forexample, by using the kinetic turbidimetric Limulus Amebocyte Lysate(LAL) assay (see Yin et al. (1972), Biochim. Biophys. Acta.261:284-289).

Inflammatory material may be assessed using a mouse peritoneal exudatecell assay which quantitates oxidative burst activity followingintraperitoneal injection of test material, e.g., as described in U.S.Pat. No. 4,780,414.

The measured specific rotation relates to the characteristicconcentration-dependent light polarization of HA in solution. At a givenconcentration, the degree of polarization at a specific wavelength is aninherent property of the molecule, characterized by its specificrotation constant [α], values of which are known from the scientificliterature (Meyer et al. (1956) Biochim. Biophys. Acta 21:506-518; Swannet al. (1968) Biochim. Biophvs. Acta 156:7-30).

Thus, HA concentration (c) may be estimated using the equation:

$c = {R \times \frac{1000}{\lbrack\alpha\rbrack}}$

where R=polarimeter reading in degrees, and c=concentration (g/L).

HA concentration can also be estimated by the colorimetric carbazoleassay (Bitter and Muir (1962) Anal. Biochem. 4:330-334), which is basedon the reaction of the carbazole reagent with the HA glucuronateresidues released upon exhaustive hydrolysis.

The molecular weight of the bulk manufactured HA is determined using thelimiting viscosity number or intrinsic viscosity (expressed in volumeper mass) obtained from viscometry measurements, and the empiricallyestablished Mark-Houwink equation, as described in Example I and in U.S.Pat. No. 4,780,414.

Absolute measurements of the HA molecular weight may be obtained usingthe low-angle laser light scattering (LALLS) method as is known in theart.

Viscosity of a biopolymer solution may be measured at discrete shearrates, e.g., using a Brookfield LVTD viscometer, as well as over acontinuous range of shear rates, e.g., using a Haake rotationalviscometer.

Upon determination that a bulk manufactured HA batch is of sufficientpurity, concentration, viscosity and molecular weight, the formulationprocess according to the method of the invention may be initiated, andall steps are preferably conducted under clean room conditions. The bulkmanufactured HA is most conveniently in solid form i.e., followingprecipitation and vacuum drying, so that using the process of theinvention it may be formulated in the desired medium, e.g., buffer andexcipients.

Preferably, the biopolymer is not subjected to freeze drying at anystage of bulk manufacturing or formulation.

An appropriate amount of bulk manufactured HA is dissolved in a suitablemedium to achieve a suitable concentration enabling sterile-filtering.

A suitable medium for biopolymer dissolution includes the buffer and/orprimary excipient found in the final formulation. For biopolymerformulations intended for injection, such a buffer or excipient shouldpreferably be physiologically acceptable. Suitable media include sodiumchloride, phosphate buffered saline, and buffers containing citrate,bicarbonate, acetate and benzylalkonium salts, including metal salts.The dissolution medium may further comprise additional excipientspresent in the final formulation such as chelating agents, isotonicityagents, antimicrobial agents, antiviral agents, preservatives andsurfactants. The dissolution medium may further comprise additionalpharmaceutically active agents such as antibiotics, antimicrobialagents, antiviral agents, steroids, non-steroidal anti-inflammatorydrugs, glucocorticoids, growth factors, prostaglandins, vitamins,enzymes, enzyme inhibitors, antioxidants, antihistamines, prodrugs,anaesthetic agents, analgesic agents, antihypertensive agents andantiangiogenic agents.

For biopolymer dissolution, an appropriate vessel is filled with waterfor injection (WFI) (70-80% of final volume) having a temperature of4-50° C. The appropriate amounts of medium reagents (dry or liquid) areadded and stirred, following which an appropriate amount of bulkmanufactured biopolymer is added. WFI is added to the appropriate finalvolume to achieve a solution in which the biopolymer concentrationpermits subsequent sterile filtration. Stirring process is carried outuntil complete dissolution of the biopolymer is achieved, typically10-36 hours for HA.

Optionally, excipients may be added towards the end of the stirringperiod, for example to avoid excess foaming of surfactants.

An appropriate vessel for dissolution is fitted with a mixing apparatussuch as a double spiral and has an industrial scale volume capacity. Thevessel should be closed to protect the biopolymer from natural andartificial illumination, thereby avoiding photo flux effects. Similarly,the material of the dissolution vessel should be inert towards thebiopolymer.

The concentration achieved by the dissolution process should be one thatenables sterile-filtration at an industrially acceptable flow rate(e.g., 750 mL/min) using an exerted pressure {e.g., 10-15 psi) whichdoes not adversely affect the molecular weight of the biopolymer.

For HA of molecular weight about 3×10⁶ daltons, an appropriateconcentration following dissolution is 0.1-0.13%; 1.0-1.3 mg/mL.

According to the method of the invention, biopolymer dissolution isfollowed by sterile filtration using an appropriate filter housed in asterilization unit. Preferably the dissolution vessel and thesterilization unit are physically connected by an appropriate tubing andvalve system which is removable, modular and sterilizable. Thedissolution and sterilization units may be located in separate roomswith the connecting tubing and valve system being positioned through thewall separating the rooms. The sterilization unit should be fitted withmeans to perform sterilization in place (SIP) and cleaning in place(CIP) of relevant components such as inlet and outlet valves.

The filter in the sterilization unit should have an absolute pore sizeof 0.05-0.2 μm and should be validated for bacterial and viral particleretention using appropriate challenge regimens. The filter is alsopreferably graded for endotoxin particle retention. The filter may behydrophilic or hydrophobic and should be selected on the basis of thehydrophobicity and charge of the biopolymer. Materials used forsterilizing filters include, but are not limited to, polyethersulfone(PES), polyvinylidene fluoride (PVDF), polytetrafluorethylene (PTFE),polypropylene, polyethylene, polyamide, cellulose, cellulose acetate,cellulose mixed esters or other cellulose derivatives and nylon.Manufacturers of suitable sterilization filters include but are notlimited to Millipore, Meissner, Sartorius, and the like.

The filter is preferably housed in a cartridge designed for industrialpurposes. Such cartridges are usually supplied in various lengths, mostcommonly 10, 20 and 30 inches, and accordingly provide differingfiltration surface areas. For example, a 30 inch Durapore™ 0.2 μm(Millipore) filter cartridge provides a surface area of 20,700 cm².

In a typical sterilization process of a solution of 0.1% NaHA, ahydrophilic 0.2 μm filter cartridge 30 inches in length, for exampleDurapore™ (Millipore) or Sartobran™ (Sartorius) is used. Filtration isperformed under 10-15 psi, and back pressure of 1.5-2 bar is appliedafter each 15-20 L of solution. A minimal flow rate of about 750 mL/minshould be maintained. The filters should be subjected to bubble pointand diffusion testing before and after each use. The filters may bereused following cleaning and sterilization according to themanufacturer's recommendation and validation of the sterilizingproperties of the re-used filters.

The sterile filtered biopolymer is fed into a concentration unit whichis connected to the sterilization unit via an appropriate tubing andvalve system. All inlet and outlet points of the concentration unitshould be fitted with means to perform SIP and CIP of relevantcomponents.

The concentration unit is fitted with an ultrafiltration membrane. Theultrafiltration membrane may be a ceramic, polysulfone,polyethersulfone, cellulose acetate, hydrolyzed PES or PVDF or stainlesssteel membrane. The ultrafiltration membrane may be of plate and frame,hollow fiber or spiral wound construction. A suitable ceramic membranemay be composed of titanium oxide, zirconium oxide, aluminum oxide,silicon oxide or mixtures thereof. The ultrafiltration membrane shouldhave a pore size of 0.002 to 0.1 μm; and a pore size of 50 nm ispreferable for HA.

Concentration by ultrafiltration is continued until the desired finalconcentration of the biopolymer is achieved. For example, a desiredfinal concentration for NaHA is 1.0-2.0%; 10-20g/L. The desired finalconcentration can be in the range from 0.8 to 3.0% w/v. The desiredfinal concentration can be about 1.0% w/v. The desired finalconcentration can be about 1.2% w/v. The desired final concentration canbe about 2.0% w/v. The desired final concentration can be in the rangefrom 0.9 to 1.3% w/v.

The efficiency of the ultrafiltration process may be assessed bydetermining the concentrations of the biopolymer in the retentate ascompared to the ultrafiltrate. A minimal concentration in theultrafiltrate, e.g., less than 1% compared to that in the retentate,indicates an acceptable level of efficiency.

Following concentration to the desired concentration, the biopolymersolution may be optionally transferred to an intermediate tank in whichdegassing and stirring are performed to ensure uniformity of theproduct.

The final formulated biopolymer product is then transferred to asuitable automated filling machine in which uniform aliquots, e.g., 0.5mL, 1.0 mL or 2.0 mL are used to fill units of a suitable sterilepackage or delivery device such as a vial, syringe, catheter ornebulizer.

The formulated biopolymer should be assessed for key quality parameters,particularly molecular weight, concentration, viscosity, osmolality,purity, endotoxin content, absorbance, pH and bioburden, as is carriedout for assessment of the bulk manufactured biopolymer. Additionalparameters associated with the final formulated product, such assyringeability and package integrity are also assessed.

In some embodiments, the formulated biopolymer, e.g., hyaluronic acid,may further comprise additional pharmaceutically active agents such asantibiotics, antimicrobial agents, antiviral agents, steroids,non-steroidal anti-inflammatory drugs, glucocorticoids, growth factors,prostaglandins, vitamins, enzymes, enzyme inhibitors, antioxidants,antihistamines, prodrugs, anaesthetic agents, analgesic agents,antihypertensive agents and antiangiogenic agents. The formulatedbiopolymer, e.g., hyaluronic acid, may also include additional compoundsfor improving joint lubrication such as a microalgal polysaccharide suchas a polysaccharide isolated from a microalga of the genus Porphyridium.Including a polysaccharide isolated from a microalga of the genusPorphyridium in a hyaluronic acid-containing formulation can providelonger half-life to the formulated hyaluronic acid due to potentinhibition of hyaluronidase by polysaccharides isolated from microalgaeof the genus Porphyridium.

Thus, the present application provides:

¶1. A process for formulating a soluble viscoelastic biopolymercomprising:

-   -   (i) sterile-filtering soluble bulk manufactured biopolymer by        passage through a membrane suitable for sterile filtration; and    -   (ii) concentrating the biopolymer by ultrafiltration to a        desired final concentration.

¶2. The process as described in ¶1, wherein the biopolymer is selectedfrom the group consisting of a homopolysaccharide, aheteropolysaccharide and mixtures thereof.

¶3. The process as described in ¶2, wherein the homopolysaccharide isselected from the group consisting of carboxymethylcellulose, chitin,polymannuronic acid, curdlan gum and dextran.

¶4. The process as described in ¶2, wherein the heteropolysaccharide isselected from the group consisting of hyaluronic acid, chondroitinsulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate,agar, alginate, carrageenan, gellan, guar gum, locust bean gum, andxanthan gum.

¶5. The process as described in ¶1, wherein the biopolymer is obtainedfrom a source selected from the group consisting of a biological source,an in vitro enzymatic synthesis, a chemical synthesis, and combinationsof two or more such sources.

¶6. The process as described in ¶5, wherein the biological source isselected from the group consisting of a bacterium, a yeast, a plant, anamphibian, an avian and a mammal.

¶7. The process as described in ¶5, wherein the biopolymer obtained froma biological source further comprises a chemical modification.

¶8. The process as described in ¶7, wherein the chemical modificationcomprises a modification selected from the group consisting of additionof sulfate groups, addition of carboxyl groups, addition of hydroxylgroups, addition of acetyl groups, esterification and cross-linking.

¶9. The process as described in ¶1, wherein the viscoelastic biopolymerhas an average molecular weight in the range from 1×10⁴ to 1×10⁷daltons.

¶10. The process as described in ¶9, wherein the viscoelastic biopolymerhas an average molecular weight of 3×10⁶±0.6×10⁶ daltons.

¶11. The process as described in ¶9, wherein the viscoelastic biopolymerhas an average molecular weight in the range from 2.8×10⁶ to 3.2×10⁶daltons.

¶12. The process as described in ¶6, wherein the bacterium is a strainof the genus Streptococcus.

¶13. The process as described in ¶12, wherein the bacterium is aStreptococcus species selected from the group consisting ofStreptococcus equi, Streptococcus pyogenes, Streptococcus equisimilis,Streptococcus dysgalactiae and Streptococcus zooepidemicus.

¶14. The process as described in ¶12, wherein the Streptococcus strainis non-hemolytic and non-pathogenic.

¶15. The process as described in ¶1, wherein the bulk manufacturedbiopolymer is isolated from a culture broth of a fermented Streptococcusstrain.

¶16. The process as described in ¶15, wherein the bulk manufacturedbiopolymer is hyaluronic acid.

¶17. The process as described in ¶16, wherein the bulk manufacturedhyaluronic acid is substantially free of impurities.

¶18. The process as described in ¶17, wherein the bulk manufacturedhyaluronic acid is substantially free of bacterial endotoxin.

¶19. The process as described in ¶18, wherein the level of bacterialendotoxin is <0.25 EU/mL.

¶20. The process as described in ¶17, wherein the bulk manufacturedhyaluronic acid is substantially free of bacterial cells.

¶21. The process as described in ¶20, wherein the viable count ofbacterial cells is <100 CFU/g.

¶22. The process as described in ¶21, wherein the viable count ofbacterial cells is <50 CFU/g.

¶23. The process as described in ¶22, wherein the viable count ofbacterial cells is <10 CFU/g.

¶24. The process as described in ¶17, wherein the bulk manufacturedhyaluronic acid is substantially free of protein.

¶25. The process as described in ¶24, wherein the level of protein is <1mg/g.

¶26. The process as described in wherein the concentration of thesoluble bulk manufactured biopolymer in step (i) is <0.2%.

¶27. The process as described in ¶26, wherein the concentration of thesoluble bulk manufactured biopolymer in step (i) is 0.10-0.13%.

¶28. The process as described in wherein the concentrating is carriedout by ultrafiltration.

¶29. The process as described in ¶28, wherein the ultrafiltration iscarried out using a ceramic membrane.

¶30. The process as described in ¶1, wherein the desired finalconcentration in step (ii) is in the range of 0.8 to 3.0% w/v.

¶31. A process as described in ¶30, wherein the desired finalconcentration is about 1.0% w/v.

¶32. A process as described in ¶30, wherein the desired finalconcentration is about 1.2% w/v.

¶33. A process as described in ¶30, wherein the desired finalconcentration is about 2.0% w/v.

¶34. A process as described in ¶30, wherein the desired finalconcentration in step (ii) is in the range from 0.9 to 1.3% w/v.

¶35. A process as described in ¶1, further comprising aseptic filling ofa suitable packaging device with the biopolymer.

¶36. A process as described in ¶35, wherein the packaging device isselected from the group consisting of a syringe, a vial, a catheter anda nebulizer.

¶37. A process as described in ¶1, wherein the formulated viscoelasticbiopolymer has a pseudoplasticity index in the range from 500 to 4000.

¶38. A process as described in ¶37, wherein the pseudoplasticity indexis in the range from 600 to 1200.

¶39. A process as described in ¶38, wherein the pseudoplasticity indexis in the range from 600 to 800.

¶40. A process as described in ¶1, wherein the sterile-filtering iscarried out using a membrane of absolute pore size 0.2 micron.

¶41. A process for formulating a viscoelastic biopolymer comprising:

-   -   (i) dissolving bulk manufactured biopolymer in a suitable buffer        medium to achieve a dilute concentration for sterile-filtering;    -   (ii) sterile-filtering the biopolymer by passage through a        membrane suitable for sterile filtration; and    -   (iii) concentrating the biopolymer by ultrafiltration to a        desired final concentration.

¶42. The process as described in ¶41, wherein the biopolymer is selectedfrom the group consisting of a homopolysaccharide, aheteropolysaccharide and mixtures thereof.

¶43. The process as described in ¶42, wherein the homopolysaccharide isselected from the group consisting of carboxymethylcellulose, chitin,polymannuronic acid, curdlan gum and dextran.

¶44. The process as described in ¶42, wherein the heteropolysaccharideis selected from the group consisting of hyaluronic acid, chondroitinsulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate,agar, alginate, carrageenan, gellan, guar gum, locust bean gum, andxanthan gum.

¶45. The process as described in ¶41, wherein the biopolymer is obtainedfrom a source selected from the group consisting of a biological source,an in vitro enzymatic synthesis, a chemical synthesis, and combinationsof two or more such sources.

¶46. The process as described in ¶45, wherein the biological source isselected from the group consisting of a bacterium, a plant, anamphibian, an avian and a mammal.

¶47. The process as described in ¶45, wherein the biopolymer obtainedfrom a biological source further comprises a chemical modification.

¶48. The process as described in ¶47, wherein the chemical modificationcomprises a modification selected from the group consisting of additionof sulfate groups, addition of carboxyl groups, addition of hydroxylgroups, addition of acetyl groups, esterification, and cross-linking.

¶49. The process as described in ¶41, wherein the viscoelasticbiopolymer has an average molecular weight in the range from 1×10⁴ to1×10⁷ daltons.

¶50. The process as described in ¶49, wherein the viscoelasticbiopolymer has an average molecular weight of 3×10⁶±0.6×10⁶ daltons.

¶51. The process as described in ¶49, wherein the viscoelasticbiopolymer has an average molecular weight in the range from 2.8×10⁶ to3.2×10⁶ daltons.

¶52. The process as described in ¶46, wherein the bacterium is a strainof the genus Streptococcus.

¶53. The process as described in ¶52, wherein the bacterium is aStreptococcus species selected from the group consisting ofStreptococcus equi, Streptococcus pyogenes, Streptococcus equisimilis,Streptococcus dysgalactiae and Streptococcus zooepidemicus.

¶54. The process as described in ¶52, wherein the Streptococcus strainis nonhemolytic and non-pathogenic.

¶55. The process as described in ¶41, wherein the bulk manufacturedbiopolymer is isolated from a culture broth of a fermented Streptococcusstrain.

¶56. The process as described in ¶55, wherein the bulk manufacturedbiopolymer is hyaluronic acid.

¶57. The process as described in ¶56, wherein the bulk manufacturedhyaluronic acid is substantially free of impurities.

¶58. The process as described in ¶57, wherein the bulk manufacturedhyaluronic acid is substantially free of bacterial endotoxin.

¶59. The process as described in ¶58, wherein the level of bacterialendotoxin is <0.25 EU/mL.

¶60. The process as described in ¶56, wherein the bulk manufacturedhyaluronic acid is substantially free of bacterial cells.

¶61. The process as described in ¶60, wherein the viable count ofbacterial cells is <100 CFU/g.

¶62. The process as described in ¶61, wherein the viable count ofbacterial cells is <50 CFU/g.

¶63. The process as described in ¶62, wherein the viable count ofbacterial cells is <10 CFU/g.

¶64. The process as described in ¶57, wherein the bulk manufacturedhyaluronic acid is substantially free of protein.

¶65. The process as described in ¶64, wherein the level of protein is <1mg/g.

¶66. The process as described in ¶41, wherein the dissolving in step (i)yields soluble bulk manufactured biopolymer at a concentration of <0.2%.

¶67. The process as described in ¶66, wherein the dissolving in step (i)yields soluble bulk manufactured biopolymer at a concentration is in therange of 0.10-0.13%.

¶68. The process as described in ¶41, wherein the concentrating in step(ii) is carried out by ultrafiltration.

¶69. The process as described in ¶68, wherein the ultrafiltration iscarried out using a ceramic membrane.

¶70. The process as described in ¶41, wherein the desired finalconcentration in step (iii) is in the range of 0.8 to 3.0% w/v.

¶71. A process as described in ¶70, wherein the desired finalconcentration is about 1.0% w/v.

¶72. A process as described in ¶70, wherein the desired finalconcentration is about 1.2% w/v.

¶73. A process as described in ¶70, wherein the desired finalconcentration is about 2.0% w/v.

¶74. A process as described in ¶41, wherein the desired finalconcentration in step (iii) is in the range from 0.9 to 1.3% w/v.

¶75. The process as described in ¶41, further comprising aseptic fillingof a suitable packaging device with the biopolymer.

¶76. The process as described in ¶75, wherein the packaging device isselected from the group consisting of a syringe, a vial, a catheter anda nebulizer.

¶77. The process as described in ¶41, wherein the formulatedviscoelastic biopolymer has a pseudoplasticity index in the range from500 to 4000.

¶78. The process as described in ¶77, wherein the pseudoplasticity indexis in the range from 600 to 1200.

¶79. The process as described in ¶78, wherein the pseudoplasticity indexis in the range from 600 to 800.

¶80. The process as described in ¶41, wherein the sterile-filtering iscarried out using a membrane of absolute pore size 0.2 micron.

¶81. The process as described in ¶41, wherein the buffer mediumcomprises a metal salt.

¶82. A process for formulating a viscoelastic preparation of hyaluronicacid comprising:

-   -   (i) dissolving bulk manufactured hyaluronic acid in a suitable        buffer medium to achieve a dilute concentration for        sterile-filtering;    -   (ii) sterile-filtering the dissolved hyaluronic acid by passage        through a 0.2 micron absolute membrane; and    -   (iii) concentrating the hyaluronic acid by ultrafiltration to a        desired final concentration.

¶83. The process as described in ¶82, wherein the bulk manufacturedhyaluronic acid is obtained from a source selected from the groupconsisting of a biological source, an in vitro enzymatic synthesis, achemical synthesis, and combinations of two or more such sources.

¶84. The process as described in ¶83, wherein the biological source isselected from the group consisting of a bacterium, a yeast, a plant, anamphibian, an avian and a mammal.

¶85. The process as described in ¶84, wherein the bulk manufacturedhyaluronic acid obtained from a biological source further comprises achemical modification.

¶86. The process as described in ¶85, wherein the chemical modificationcomprises a modification selected from the group consisting of additionof sulfate groups, addition of carboxyl groups, addition of hydroxylgroups, addition of acetyl groups, esterification, and cross-linking.

¶87. The process as described in ¶82, wherein the bulk manufacturedhyaluronic acid has an average molecular weight in the range from 1×10⁴to 1×10⁷ daltons.

¶88. The process as described in ¶87, wherein the bulk manufacturedhyaluronic acid has an average molecular weight of 3×10⁶±0.6×10⁶daltons.

¶89. The process as described in ¶87, wherein the bulk manufacturedhyaluronic acid has an average molecular weight in the range from2.8×10⁶ to 3.2×10⁶ daltons.

¶90. The process as described in ¶84, wherein the bacterium is a speciesof the genus Streptococcus.

¶91. The process as described in ¶90, wherein the bacterium is aStreptococcus species selected from the group consisting ofStreptococcus equi, Streptococcus pyogenes, Streptococcus equisimilis,Streptococcus dysgalactiae and Streptococcus zooepidemicus.

¶92. The process as described in ¶90, wherein the Streptococcus strainis non-hemolytic and non-pathogenic.

¶93. The process as described in ¶83, wherein the bulk manufacturedhyaluronic acid is isolated from a culture broth of a fermentedStreptococcus strain.

¶94. The process as described in ¶82, wherein the bulk manufacturedhyaluronic acid is substantially free of impurities.

¶95. The process as described in ¶94, wherein the bulk manufacturedhyaluronic acid is substantially free of bacterial endotoxin.

¶96. The process as described in ¶95, wherein the level of bacterialendotoxin is <0.25 EU/mL.

¶97. The process as described in ¶94, wherein the bulk manufacturedhyaluronic acid is substantially free of bacterial cells.

¶98. The process as described in ¶97, wherein the viable count ofbacterial cells is <100 CFU/g.

¶99. The process as described in ¶98, wherein the viable count ofbacterial cells is <50 CFU/g.

¶100. The process as described in ¶99, wherein the viable count ofbacterial cells is <10 CFU/g.

¶101. The process as described in ¶94, wherein the bulk manufacturedhyaluronic acid is substantially free of protein.

¶102. The process as described in ¶101, wherein the level of protein is<1 mg/g.

¶103. The process as described in ¶82, wherein the concentration of thedissolved bulk manufactured biopolymer obtained in step (i) is <0.2%.

¶104. The process as described in ¶103, wherein the concentration of thedissolved bulk manufactured biopolymer obtained in step (i) is0.10-0.13%.

¶105. The process as described in ¶82, wherein the concentrating iscarried out by ultrafiltration.

¶106. The process as described in ¶105, wherein the ultrafiltration iscarried out using a ceramic membrane.

¶107. The process as described in ¶82, wherein the desired finalconcentration in step (iii) is in the range of 0.8 to 3.0% w/v.

¶108. A process as described in ¶107, wherein the desired finalconcentration is about 1.0% w/v.

¶109. A process as described in ¶107, wherein the desired finalconcentration is about 1.2% w/v.

¶110. A process as described in ¶107, wherein the desired finalconcentration is about 2.0% w/v.

¶111. A process as described in ¶82, wherein the desired finalconcentration in step (iii) is in the range from 0.9 to 1.3% w/v.

¶112. A process as described in ¶82, further comprising aseptic fillingof a suitable packaging device with the biopolymer.

¶113. A process as described in ¶112, wherein the packaging device isselected from the group consisting of a syringe, a vial, a catheter anda nebulizer.

¶114. A process as described in ¶82, wherein the formulated hyaluronicacid has a pseudoplasticity index in the range from 500 to 4000.

¶115. A process as described in ¶114, wherein the pseudoplasticity indexis in the range from 600 to 1200.

¶116. A process as described in ¶115, wherein the pseudoplasticity indexis in the range from 600 to 800.

¶117. A process as described in ¶82, wherein the sterile-filtering iscarried out using a membrane of absolute pore size 0.2 micron.

¶118. A process as described in ¶82, wherein all steps are performedunder clean room conditions.

¶119. A process as described in ¶82, wherein the bulk manufacturedhyaluronic acid is obtained by a process comprising:

-   -   (i) precipitating with ethanol a culture broth of a        non-hemolytic nonpathogenic hyaluronic acid-producing fermented        Streptococcus strain;    -   (ii) dissolving the precipitate obtained in step (i) in sodium        chloride/ethanol/charcoal;    -   (iii) precipitating the dissolved material obtained in step (ii)        with cetylpyridinium chloride;    -   (iv) dissolving the precipitate obtained in step (iii) in sodium        chloride/ethanol;    -   (v) treating the dissolved material obtained in step (iv) with        magnesium silicate;    -   (vi) filtering the treated material obtained in step (v) through        a 0.65 micron absolute membrane; and    -   (vii) precipitating the filtrate obtained in step (vi) with        ethanol.

¶120. A formulation of viscoelastic hyaluronic acid suitable forinjection during surgery to mammals, obtained by the process asdescribed in ¶82.

¶121. The formulation as described in ¶120, substantially free ofimpurities and having a pseudoplasticity index greater than 600.

¶122. The formulation as described in ¶121, wherein the hyaluronic acidhas an average molecular weight of 3×10⁶±0.6×10⁶ daltons.

¶123. The process as described in ¶121, wherein the hyaluronic acid hasan average molecular weight in the range from 2.8×10⁶ to 3.2×10⁶daltons.

¶124. The formulation as described in ¶120, further comprising a drug.

¶125. The formulation as described in ¶120, wherein the hyaluronic acidis chemically cross-linked.

¶126. The formulation as described in ¶120, wherein the hyaluronic acidis complexed with a metal.

¶126. The formulation as described in ¶120, further comprising amicroalgal polysaccharide.

¶127. The formulation as described in ¶126, wherein the microalgalpolysaccharide is a polysaccharide isolated from a Porphyridiummicroalga.

¶127. The process as described in any of ¶1, ¶41 or ¶82, wherein theresulting viscoelastic biopolymer is stable and sterile for at leastabout one year.

¶128. The process of claim 120, wherein the resulting viscoelasticbiopolymer is stable and sterile for at least about two years.

¶129. The process of claim 120, wherein the resulting viscoelasticbiopolymer is stable and sterile for at least about five years.

¶130. The process as described in any of ¶1, ¶41 or ¶82, wherein nopreservative of the viscoelastic biopolymer is used.

¶131. The process as described in any of ¶1, ¶41 or ¶182, furthercomprising including a microalgal polysaccharide in the formulation.

¶132. The process as described in ¶131, wherein the microalgalpolysaccharide is a polysaccharide isolated from a Porphyridiummicroalga.

All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference in their entirety.

What is claimed is:
 1. A process for formulating hyaluronic acid, theprocess comprising: (i) dissolving bulk-manufactured hyaluronic acidhaving a high molecular weight of 3.0±0.6 megadaltons in an aqueousmedium to form a hyaluronic acid solution having a concentration of lessthan 0.2%; (ii) sterile-filtering the hyaluronic acid solution having aconcentration of less than 0.2% by passage through a membrane suitablefor sterile filtration at a pressure that does not adversely affect thehigh molecular weight of the hyaluronic acid, whereby thesterile-filtering retains the high molecular weight structure of thehyaluronic acid in the hyaluronic acid solution; and (iii) subsequentlyconcentrating the sterile-filtered hyaluronic acid by ultrafiltrationwith a membrane having a pore size of 0.002 to 0.1 microns under asepticconditions to form a sterile solution formulation of a desired finalconcentration; wherein the formulation is suitable for medicinal use byinjection into a human without further purification following step(iii); and wherein the concentrated hyaluronic acid in the sterilesolution formulation has a high molecular weight of 3.0±0.6 megadaltons.2. The process according to claim 1, wherein the hyaluronic acid has anaverage molecular weight in the range from 2.8×10⁶ to 3.2×10⁶ daltons.3. The process according to claim 1, further comprising obtaining thebulk manufactured hyaluronic acid for step (i) by isolation of thehyaluronic acid from a culture broth of a fermented Streptococcusstrain.
 4. The process according to claim 3, wherein the Streptococcusis a Streptococcus species selected from the group consisting ofStreptococcus equi, Streptococcus pyogenes, Streptococcus equisimilis,Streptococcus dysgalactiae and Streptococcus zooepidemicus or anotherStreptococcus strain that is non-hemolytic and non-pathogenic.
 5. Theprocess according to claim 1, wherein the bulk manufactured hyaluronicacid is substantially free of impurities.
 6. The process according toclaim 1, wherein the concentration of the hyaluronic acid in step (i) is0.10-0.13% w/v.
 7. The process according to claim 1, wherein themembrane suitable for sterile filtration has a pore size of 0.05 to 0.2microns.
 8. The process according to claim 1, wherein the concentratingis carried out by ultrafiltration using a ceramic membrane.
 9. Theprocess according to claim 8, wherein the ultrafiltration is carried outwith a membrane having a pore size of about 50 nm.
 10. The processaccording to claim 1, wherein the desired final concentration in step(iii) is in the range from 0.8 to 3.0% w/v.
 11. The process according toclaim 9, wherein the desired final concentration in step (iii) is about1.0% w/v to about 2.0% w/v.
 12. The process according to claim 9,wherein the desired final concentration in step (iii) is in the rangefrom 0.9 to 1.3% w/v.
 13. The process according to claim 1, furthercomprising aseptically filling a suitable packaging device with thehyaluronic acid solution obtained in step (iii).
 14. The processaccording to claim 13, wherein the packaging device is selected from thegroup consisting of a syringe, a vial, a catheter, and a nebulizer. 15.The process according to claim 1, wherein the formulated hyaluronic acidhas a pseudoplasticity index in the range from 500 to
 4000. 16. Theprocess according to claim 1, further comprising including a microalgalpolysaccharide in the formulation.
 17. The process according to claim16, wherein the microalgal polysaccharide is a polysaccharide isolatedfrom a Porphyridium microalga.
 18. The process according to claim 1,wherein all steps are performed under clean room conditions.
 19. Theprocess according to claim 1, wherein the hyaluronic acid is notsubjected to freeze drying at any stage of bulk manufacturing orformulation.